Q.1 What is the primary function of a plasmid vector in cloning?
To replicate independently in a host cell
To inhibit DNA replication
To degrade foreign DNA
To serve as a template for protein synthesis
Explanation - A plasmid vector carries an origin of replication that allows it to replicate autonomously within a host bacterium, thereby maintaining the inserted gene.
Correct answer is: To replicate independently in a host cell
Q.2 Which antibiotic resistance gene is most commonly used as a selectable marker in bacterial cloning?
kanamycin
chloramphenicol
ampicillin
streptomycin
Explanation - Ampicillin resistance conferred by the beta-lactamase gene (bla) is widely used because many cloning strains lack this resistance and can be selected against.
Correct answer is: ampicillin
Q.3 A multiple cloning site (MCS) is characterized by:
a single restriction site
a sequence of tandem repeats of the same base
multiple unique restriction enzyme sites
a region coding for fluorescent proteins
Explanation - An MCS contains a series of unique restriction sites that allow flexibility in inserting foreign DNA using various enzymes.
Correct answer is: multiple unique restriction enzyme sites
Q.4 Which enzyme is responsible for sealing nicks in DNA after ligation?
DNA polymerase
DNA ligase
DNA helicase
DNA methyltransferase
Explanation - DNA ligase catalyzes the formation of a phosphodiester bond between adjacent nucleotides, closing the nicks in a newly synthesized strand.
Correct answer is: DNA ligase
Q.5 Which of the following is NOT a typical feature of a eukaryotic plasmid vector?
Origin of replication
Selectable marker
Promoter for eukaryotic expression
PolyA tail signal
Explanation - The polyadenylation signal is part of the mRNA, not a feature of the plasmid vector itself.
Correct answer is: PolyA tail signal
Q.6 What is the role of a 'promoter' in a cloning vector?
To provide antibiotic resistance
To initiate transcription of the inserted gene
To prevent recombination
To create a fluorescent tag
Explanation - A promoter is a DNA sequence upstream of the gene that recruits RNA polymerase to start transcription.
Correct answer is: To initiate transcription of the inserted gene
Q.7 Which cloning technique uses a temperature-sensitive origin of replication?
Red/ET recombination
Gateway cloning
Temperature-sensitive plasmid cloning
Yeast two-hybrid system
Explanation - Temperature-sensitive plasmids replicate at permissive temperatures but cannot replicate at higher temperatures, facilitating plasmid curing.
Correct answer is: Temperature-sensitive plasmid cloning
Q.8 Which enzyme is commonly used to generate blunt-ended DNA fragments?
SmaI
EcoRI
Taq DNA polymerase
AluI
Explanation - SmaI cuts with a 3' overhang that is then processed to yield blunt ends, making it useful for blunt-end cloning.
Correct answer is: SmaI
Q.9 The 'lacZα' gene fragment in a plasmid vector is used for:
Blue-white screening
Antibiotic resistance
Selection of eukaryotic hosts
mRNA stability
Explanation - Insertion of foreign DNA into the lacZα gene disrupts β-galactosidase activity, allowing blue-white screening on X-gal plates.
Correct answer is: Blue-white screening
Q.10 Which cloning strategy relies on site-specific recombination mediated by the λ phage integrase?
Gateway cloning
Restriction enzyme cloning
TALENs
CRISPR-Cas9
Explanation - Gateway cloning uses attP, attB, attL, and attR sites and integrase enzymes from λ phage for efficient recombination.
Correct answer is: Gateway cloning
Q.11 In Gibson Assembly, which enzyme is NOT required?
Exonuclease
DNA polymerase
DNA ligase
RNA polymerase
Explanation - Gibson Assembly uses exonuclease, polymerase, and ligase to join DNA fragments with overlapping ends.
Correct answer is: RNA polymerase
Q.12 What is the main advantage of using a BAC (bacterial artificial chromosome) vector?
It can clone very large DNA fragments (~300 kb)
It is inexpensive to produce
It confers resistance to all antibiotics
It has a high copy number in mammalian cells
Explanation - BACs are designed to stably maintain large inserts, making them suitable for cloning whole genes or genomic regions.
Correct answer is: It can clone very large DNA fragments (~300 kb)
Q.13 Which of the following is NOT a feature of a YAC (yeast artificial chromosome)?
Contains a yeast centromere
Can carry up to 1 Mb of DNA
Has a yeast telomere sequence
Requires bacterial replication origin
Explanation - YACs replicate in yeast using yeast origins of replication, not bacterial ones.
Correct answer is: Requires bacterial replication origin
Q.14 What is the purpose of a 'vector backbone'?
To provide the antibiotic resistance gene
To carry the gene of interest
To provide origin of replication and other regulatory elements
To produce proteins in the host
Explanation - The backbone contains the essential elements for the vector's maintenance and function.
Correct answer is: To provide origin of replication and other regulatory elements
Q.15 Which restriction enzyme generates 5' overhangs of 4 nucleotides?
EcoRI
BamHI
NotI
HindIII
Explanation - EcoRI cuts between G and A producing 5' overhangs of 4 bases (5'-AATT-3').
Correct answer is: EcoRI
Q.16 Why is a 'negative selection' marker used in plasmid cloning?
To select for cells that have lost the plasmid
To kill cells that have the plasmid
To provide an alternative antibiotic resistance
To ensure plasmids are high copy number
Explanation - Negative selection markers (e.g., sacB) kill cells that retain the plasmid under certain conditions, aiding plasmid curing.
Correct answer is: To kill cells that have the plasmid
Q.17 What is the function of a 'selection marker' in a cloning vector?
To allow the vector to replicate in the host
To ensure expression of the inserted gene
To confer resistance to an antibiotic
To provide a fluorescent tag
Explanation - Selection markers enable growth of only those cells that have incorporated the vector.
Correct answer is: To confer resistance to an antibiotic
Q.18 Which cloning method uses a 'stuffer' DNA fragment that is replaced by the gene of interest?
TOPO cloning
Restriction cloning
Ligase cycling reaction
Stuffer replacement cloning
Explanation - Stuffer replacement cloning involves a filler DNA that is cut and replaced by the target insert, often using recombination or ligation.
Correct answer is: Stuffer replacement cloning
Q.19 Which technique is used to insert a gene into a plasmid using PCR amplicons with overlapping ends?
Restriction digestion and ligation
Golden Gate Assembly
Inverse PCR
Overlap PCR cloning
Explanation - Overlap PCR allows the generation of overlapping ends that can anneal and be ligated into a plasmid backbone.
Correct answer is: Overlap PCR cloning
Q.20 Which feature is essential for a plasmid to be considered a 'high copy number' vector?
Large size
Strong promoter
Strong origin of replication
Multiple antibiotic resistance genes
Explanation - A strong, efficient origin of replication drives many copies of the plasmid in each host cell.
Correct answer is: Strong origin of replication
Q.21 What does 'colicin' refer to in the context of plasmid vectors?
An antibiotic used for selection
A plasmid-encoded toxin that kills competitor bacteria
A fluorescent protein marker
A restriction enzyme
Explanation - Colicin genes encode bacteriocins that inhibit growth of closely related bacterial strains.
Correct answer is: A plasmid-encoded toxin that kills competitor bacteria
Q.22 In a ligation reaction, why is it important to keep the molar ratio of insert to vector low?
To reduce background colonies from self-ligated vector
To increase the number of colonies
To prevent restriction enzyme activity
To ensure equal copy numbers in cells
Explanation - A low insert-to-vector ratio favors correct ligation events and reduces self-ligation of the vector.
Correct answer is: To reduce background colonies from self-ligated vector
Q.23 Which enzyme is required for the 'inverse PCR' method?
T4 DNA ligase
DNA polymerase I
DNA polymerase II
RNA polymerase
Explanation - Inverse PCR amplifies across a known region in the opposite direction, requiring a high-fidelity DNA polymerase.
Correct answer is: DNA polymerase I
Q.24 Which of the following is a hallmark of a suicide vector?
It can replicate in the host but not in the donor
It has a broad-host-range origin of replication
It contains a reporter gene
It can be recovered from the host
Explanation - A suicide vector cannot replicate in the donor cell, ensuring it is transferred only into a new host.
Correct answer is: It can replicate in the host but not in the donor
Q.25 What is the role of the 'origin of transfer (oriT)' in conjugative plasmids?
To initiate replication
To allow plasmid transfer between cells
To confer antibiotic resistance
To code for a fluorescent protein
Explanation - oriT is the site where the conjugation machinery initiates plasmid transfer.
Correct answer is: To allow plasmid transfer between cells
Q.26 Which cloning strategy uses a 'self-ligating' plasmid that contains a blunt-end cutter and a ligase for seamless cloning?
TOPO cloning
Gibson Assembly
Ligase Cycling Reaction
PCR-based cloning
Explanation - TOPO cloning takes advantage of topoisomerase I to ligate PCR products without the need for restriction enzymes or ligase.
Correct answer is: TOPO cloning
Q.27 What is the main difference between a plasmid and a BAC vector?
Plasmids can replicate only in bacteria
BACs have a higher copy number
BACs can carry larger inserts (~300 kb)
Plasmids are linear DNA molecules
Explanation - BACs are engineered to stably maintain large DNA fragments, whereas typical plasmids are limited to ~10 kb.
Correct answer is: BACs can carry larger inserts (~300 kb)
Q.28 Which of the following is an example of a 'destination vector' in Gateway cloning?
pDONR207
pGEM-T Easy
pDEST32
pUC19
Explanation - Destination vectors accept the recombined product from the entry clone, enabling expression of the gene in a desired system.
Correct answer is: pDEST32
Q.29 Which technique uses homologous recombination in yeast to assemble multiple DNA fragments?
Gibson Assembly
Yeast homologous recombination
CRISPR-Cas9 editing
Molecular beacon assay
Explanation - Yeast cells efficiently recombine overlapping DNA fragments, allowing multi-fragment assembly without enzymes.
Correct answer is: Yeast homologous recombination
Q.30 Which of the following is NOT a commonly used E. coli strain for plasmid propagation?
DH5α
BL21(DE3)
TOP10
JM109
Explanation - BL21(DE3) is used for protein expression, not typically for cloning plasmids due to its protease-deficient phenotype.
Correct answer is: BL21(DE3)
Q.31 Why are 'stop codons' often omitted from cloning vectors that are designed for protein expression?
To prevent premature termination of translation
To avoid antibiotic resistance
To reduce plasmid size
To increase copy number
Explanation - Vectors designed for protein expression often lack internal stop codons so that the inserted gene can be translated correctly.
Correct answer is: To prevent premature termination of translation
Q.32 What is the purpose of a 'fusion tag' in cloning vectors?
To provide antibiotic resistance
To assist in purification of expressed proteins
To inhibit host gene expression
To increase plasmid copy number
Explanation - Fusion tags such as His6, GST, or MBP facilitate affinity purification of recombinant proteins.
Correct answer is: To assist in purification of expressed proteins
Q.33 Which enzyme is required for the creation of sticky ends during restriction digestion?
DNA polymerase
DNA ligase
Restriction endonuclease
RNA polymerase
Explanation - Restriction enzymes cut DNA at specific recognition sites, generating sticky (or blunt) ends.
Correct answer is: Restriction endonuclease
Q.34 Which cloning strategy is most suitable for inserting a large gene (over 10 kb) into a mammalian expression vector?
Restriction enzyme cloning
TOPO cloning
Red/ET recombination
Golden Gate Assembly
Explanation - Red/ET recombination in E. coli allows efficient recombination of large DNA fragments into vectors.
Correct answer is: Red/ET recombination
Q.35 In site-directed mutagenesis, which reagent is commonly used to introduce a point mutation?
DNA polymerase I
PCR primers containing the desired mutation
Taq polymerase
Restriction enzymes
Explanation - Primers designed with the mutation introduce it during PCR amplification of the plasmid.
Correct answer is: PCR primers containing the desired mutation
Q.36 Which of the following is a hallmark of a 'pUC' type plasmid?
Low copy number
High copy number
Eukaryotic promoter
No antibiotic resistance gene
Explanation - pUC vectors have a high-copy origin of replication, yielding many plasmid copies per cell.
Correct answer is: High copy number
Q.37 What is the function of a 'promoter' upstream of the multiple cloning site in a plasmid?
To facilitate antibiotic selection
To drive expression of the inserted gene
To provide a site for restriction digestion
To enable plasmid replication
Explanation - The promoter initiates transcription of the gene inserted into the MCS.
Correct answer is: To drive expression of the inserted gene
Q.38 Which cloning method utilizes 'Golden Gate' technology?
Restriction enzyme cloning
Gibson Assembly
Golden Gate Assembly
TOPO cloning
Explanation - Golden Gate uses Type IIS restriction enzymes and T4 DNA ligase to assemble DNA fragments in a single reaction.
Correct answer is: Golden Gate Assembly
Q.39 What is the main advantage of using a 'lacZ' reporter gene in plasmid vectors?
It provides antibiotic resistance
It allows blue-white screening
It enhances plasmid replication
It tags proteins with fluorescence
Explanation - The lacZ gene encodes β-galactosidase, enabling detection of successful cloning by color change on X-gal plates.
Correct answer is: It allows blue-white screening
Q.40 Which technique is used to insert a gene into a plasmid without the need for restriction enzymes?
Restriction cloning
PCR-based cloning with overlapping ends
Ligase-mediated cloning
Methylation cloning
Explanation - Overlap PCR or Gibson Assembly allows direct insertion using PCR products with compatible ends.
Correct answer is: PCR-based cloning with overlapping ends
Q.41 Which of the following plasmids is known for having an 'origin of replication' that allows very high copy numbers in E. coli?
pUC18
pBR322
pBluescript
pSC101
Explanation - pUC18 is engineered to produce thousands of copies per cell due to its modified origin.
Correct answer is: pUC18
Q.42 What is the primary purpose of a 'selection marker' that confers resistance to kanamycin?
To ensure plasmid replication
To kill cells that have not taken up the plasmid
To promote expression of the inserted gene
To enhance blue-white screening
Explanation - Kanamycin selection allows growth only of bacteria harboring the plasmid with the resistance gene.
Correct answer is: To kill cells that have not taken up the plasmid
Q.43 Which of the following is a property of a 'low-copy plasmid'?
It produces many plasmid copies per cell
It is easier to isolate plasmid DNA
It contains a strong promoter
It is often used for toxic gene expression
Explanation - Low-copy plasmids minimize gene dosage, reducing potential toxicity from overexpression.
Correct answer is: It is often used for toxic gene expression
Q.44 Why is a 'buffer' necessary during a ligation reaction?
To neutralize pH
To stabilize the DNA and ligase enzyme
To increase the concentration of antibiotics
To digest the DNA ends
Explanation - The ligation buffer provides optimal conditions (pH, ions) for ligase activity and DNA stability.
Correct answer is: To stabilize the DNA and ligase enzyme
Q.45 Which method allows direct cloning of PCR products with 'blunt ends' into a plasmid backbone?
TOPO cloning
Restriction enzyme cloning
Gibson Assembly
Golden Gate Assembly
Explanation - TOPO cloning uses topoisomerase to ligate blunt-ended PCR products without restriction digestion.
Correct answer is: TOPO cloning
Q.46 Which of the following is not a typical feature of a 'mammalian expression vector'?
CMV promoter
SV40 origin of replication
Neomycin resistance gene
Polyadenylation signal
Explanation - Mammalian vectors use viral promoters and selection markers but do not typically contain a bacterial origin of replication.
Correct answer is: SV40 origin of replication
Q.47 Which enzyme is commonly used to 'close' nicks in double-stranded DNA during cloning?
DNA ligase
Taq polymerase
RNA polymerase
DNA helicase
Explanation - DNA ligase seals the phosphodiester backbone, creating continuous DNA strands.
Correct answer is: DNA ligase
Q.48 What is a key advantage of using 'Yeast Artificial Chromosomes (YACs)' for cloning?
They replicate in bacteria
They can carry very large DNA fragments
They are easy to transform into mammalian cells
They confer antibiotic resistance
Explanation - YACs can accommodate up to 1 megabase of DNA, making them suitable for large genomic regions.
Correct answer is: They can carry very large DNA fragments
Q.49 In a cloning experiment, why might you use a 'blue-white screening' assay?
To select for antibiotic resistance
To identify colonies that have successfully incorporated the insert
To measure plasmid copy number
To ensure plasmid replication
Explanation - Blue colonies indicate intact lacZ, while white colonies indicate disruption by an insert.
Correct answer is: To identify colonies that have successfully incorporated the insert
Q.50 Which cloning method relies on the enzyme 'T4 DNA ligase' and a 'restriction enzyme' in a single step?
Gibson Assembly
TOPO cloning
Restriction enzyme cloning
Golden Gate Assembly
Explanation - Traditional restriction cloning uses restriction digestion followed by ligation with T4 DNA ligase.
Correct answer is: Restriction enzyme cloning
Q.51 What is the purpose of an 'origin of transfer (oriT)' sequence on a plasmid?
To allow replication in bacteria
To enable plasmid transfer via conjugation
To provide antibiotic selection
To encode a fluorescent protein
Explanation - oriT is the site recognized by the conjugation machinery for plasmid transfer between cells.
Correct answer is: To enable plasmid transfer via conjugation
Q.52 Which of the following is NOT an advantage of using a 'high-copy number' plasmid for protein expression?
Higher yield of recombinant protein
Increased plasmid stability
Reduced metabolic burden
Simplified plasmid purification
Explanation - High-copy plasmids often impose a higher metabolic burden on the host cell.
Correct answer is: Reduced metabolic burden
Q.53 Which cloning strategy uses a 'catalytically inactive' version of a restriction enzyme to facilitate insertion?
Golden Gate Assembly
TA cloning
In-Fusion cloning
Gibson Assembly
Explanation - In-Fusion cloning uses a proprietary enzyme that joins overlapping ends without the need for restriction digestion.
Correct answer is: In-Fusion cloning
Q.54 Which of the following describes the 'T7 promoter' in a plasmid vector?
It is a bacterial origin of replication
It initiates transcription in E. coli
It is used for high-level transcription in vitro
It provides antibiotic resistance
Explanation - The T7 promoter is recognized by T7 RNA polymerase, enabling strong in vitro transcription.
Correct answer is: It is used for high-level transcription in vitro
Q.55 Which technique is used to insert a gene into a plasmid backbone without using any restriction enzymes?
Restriction digestion
Homologous recombination in yeast
Gibson Assembly
PCR-based cloning
Explanation - Yeast can recombine DNA fragments with overlapping homology, bypassing restriction enzyme usage.
Correct answer is: Homologous recombination in yeast
Q.56 In a 'ligation-independent cloning' (LIC) strategy, what is the key enzymatic step?
Restriction digestion
Annealing of complementary overhangs
Polymerase chain reaction
Ligase activity
Explanation - LIC creates complementary single-stranded overhangs that anneal without the need for ligase.
Correct answer is: Annealing of complementary overhangs
Q.57 Which vector feature allows for selection of clones in both bacterial and yeast hosts?
E. coli origin of replication
Yeast centromere (CEN)
Dual antibiotic resistance
Fluorescent marker
Explanation - A CEN element ensures stable maintenance in yeast, while a bacterial origin allows replication in E. coli.
Correct answer is: Yeast centromere (CEN)
Q.58 What does the acronym 'Gibson Assembly' stand for?
Genome Integration By Optimized Cloning
Gluconeogenesis in Bacterial Insertion
Genome Integration By Inverse Cloning
No standard acronym; it's a method name
Explanation - Gibson Assembly is a name for a seamless cloning method, not an acronym.
Correct answer is: No standard acronym; it's a method name
Q.59 Which enzyme is required for the 'T7 RNA polymerase' transcription of a plasmid carrying a T7 promoter?
T7 RNA polymerase
T4 DNA ligase
Taq polymerase
Reverse transcriptase
Explanation - T7 RNA polymerase recognizes the T7 promoter and transcribes the downstream gene.
Correct answer is: T7 RNA polymerase
Q.60 What is a characteristic of the 'pGEM-T Easy' vector?
It contains a T7 promoter
It has a T-overhang for TA cloning
It is a high-copy plasmid
It confers kanamycin resistance
Explanation - pGEM-T Easy has 3' T overhangs that facilitate TA cloning of PCR products with 3' A overhangs.
Correct answer is: It has a T-overhang for TA cloning
Q.61 Which of the following is a common use for a 'pUC' plasmid?
Expression of viral proteins
Cloning of large genomic fragments
High-yield protein production in yeast
Molecular beacon synthesis
Explanation - pUC plasmids are frequently used for cloning due to their high copy number and large MCS.
Correct answer is: Cloning of large genomic fragments
Q.62 What is the main function of the 'lacZ α-complementation' system in cloning?
To provide antibiotic resistance
To allow blue-white screening
To produce a fluorescent protein
To ensure plasmid replication
Explanation - Disruption of lacZ α leads to white colonies on X-gal plates, indicating successful cloning.
Correct answer is: To allow blue-white screening
Q.63 Which of the following plasmids is most suitable for expressing a toxic protein in E. coli?
pUC19
pBAD24
pBR322
pGEM-T Easy
Explanation - pBAD24 has an arabinose-inducible promoter, allowing controlled expression of potentially toxic genes.
Correct answer is: pBAD24
Q.64 What is the purpose of a 'terminator sequence' in a plasmid vector?
To terminate DNA replication
To prevent read-through transcription
To confer antibiotic resistance
To enhance plasmid copy number
Explanation - A terminator ensures that RNA polymerase stops transcribing the inserted gene, preventing unwanted transcription.
Correct answer is: To prevent read-through transcription
Q.65 Which cloning method uses a 'Type IIS' restriction enzyme to create non-compatible overhangs?
Gibson Assembly
Golden Gate Assembly
TOPO cloning
Restriction enzyme cloning
Explanation - Golden Gate uses Type IIS enzymes that cut outside their recognition site, creating unique overhangs.
Correct answer is: Golden Gate Assembly
Q.66 Which of the following describes a 'vector backbone'?
The region containing the gene of interest
The minimal set of sequences required for maintenance and selection of the vector
The promoter sequence
The antibiotic resistance gene
Explanation - The backbone includes origin of replication, selectable marker, and sometimes a reporter gene.
Correct answer is: The minimal set of sequences required for maintenance and selection of the vector
Q.67 What is the role of a 'shuttle vector'?
To replicate in multiple host species
To provide fluorescence
To confer resistance to multiple antibiotics
To carry large DNA fragments
Explanation - Shuttle vectors contain origins of replication for different hosts, enabling cloning in bacteria and yeast, for example.
Correct answer is: To replicate in multiple host species
Q.68 Which technique involves the use of a 'T5 exonuclease' to create single-stranded overhangs for DNA assembly?
Gibson Assembly
TOPO cloning
Golden Gate Assembly
Restriction enzyme cloning
Explanation - Gibson Assembly uses T5 exonuclease to chew back ends, allowing annealing of overlapping fragments.
Correct answer is: Gibson Assembly
Q.69 Which of the following is a characteristic of the 'pGEX' vector family?
They carry a His-tag
They contain a GST fusion tag
They are high-copy plasmids
They are used for yeast expression
Explanation - pGEX vectors fuse the gene of interest to glutathione S-transferase for affinity purification.
Correct answer is: They contain a GST fusion tag
Q.70 What does 'Blue-white screening' detect in a cloning experiment?
Antibiotic resistance
Successful insertion disrupting lacZ
High plasmid copy number
Protein expression levels
Explanation - White colonies lack β-galactosidase activity because lacZ is disrupted by an insert.
Correct answer is: Successful insertion disrupting lacZ
Q.71 Which of the following best describes a 'colony PCR'?
PCR performed on plasmid DNA isolated from a culture
PCR directly on a bacterial colony to check for the presence of an insert
PCR to amplify plasmid vector only
PCR to insert a gene into the plasmid
Explanation - Colony PCR uses a small sample of a bacterial colony as a template for rapid screening.
Correct answer is: PCR directly on a bacterial colony to check for the presence of an insert
Q.72 Which of the following is a feature of the 'pET' vector family?
They contain a T7 promoter
They are low-copy plasmids
They lack antibiotic selection
They are used for mammalian expression
Explanation - pET vectors are designed for IPTG-inducible expression driven by a T7 promoter.
Correct answer is: They contain a T7 promoter
Q.73 Which of the following describes the 'PstI' restriction enzyme?
It generates blunt ends
It generates 5' overhangs of 4 nucleotides
It recognizes a palindromic sequence
It is a Type IIS enzyme
Explanation - PstI cuts at CTGCAG, creating 5' overhangs of 4 bases.
Correct answer is: It generates 5' overhangs of 4 nucleotides
Q.74 Which cloning strategy uses 'inverted PCR' to generate a linear fragment containing a gene of interest?
Inverse PCR
Gibson Assembly
Golden Gate Assembly
TOPO cloning
Explanation - Inverse PCR amplifies DNA in the opposite orientation, producing a linear fragment for cloning.
Correct answer is: Inverse PCR
Q.75 Which of the following is not a typical selection marker used in mammalian expression vectors?
Neomycin (G418)
Puromycin
Ampicillin
Blasticidin
Explanation - Ampicillin is used for bacterial selection; mammalian vectors use antibiotic markers like G418.
Correct answer is: Ampicillin
Q.76 What is the purpose of a 'multiple cloning site (MCS)' in a plasmid vector?
To provide a promoter
To provide antibiotic selection
To allow insertion of DNA using various restriction enzymes
To encode a fluorescent protein
Explanation - The MCS contains several unique restriction sites for flexible cloning.
Correct answer is: To allow insertion of DNA using various restriction enzymes
Q.77 Which cloning technique uses 'in silico' design of primers that introduce restriction sites for cloning?
PCR-based cloning
Golden Gate Assembly
TOPO cloning
Restriction enzyme cloning
Explanation - Primers can be designed to add restriction sites, enabling downstream cloning.
Correct answer is: PCR-based cloning
Q.78 Which plasmid backbone is known for containing the 'pUC' origin and a multiple cloning site for easy cloning?
pBR322
pUC18
pGEM-T Easy
pET-21b
Explanation - pUC18 is a high-copy plasmid with a broad MCS, widely used for cloning.
Correct answer is: pUC18
Q.79 Which feature of a plasmid vector allows it to replicate in yeast but not in bacteria?
Yeast centromere (CEN)
Bacterial origin of replication
Eukaryotic promoter
Antibiotic resistance gene
Explanation - The CEN element ensures maintenance in yeast cells, but has no function in bacteria.
Correct answer is: Yeast centromere (CEN)
Q.80 What is the role of the 'T7 gene 10 leader' sequence in pET vectors?
To enhance transcription initiation
To facilitate ribosomal binding and translation initiation
To provide antibiotic resistance
To encode a fluorescent protein
Explanation - The T7 gene 10 leader optimizes translation of the cloned gene.
Correct answer is: To facilitate ribosomal binding and translation initiation
Q.81 Which of the following is NOT an advantage of using 'Golden Gate Assembly'?
Single-tube reaction
High fidelity due to enzyme specificity
Can assemble multiple fragments in defined order
Requires linear DNA fragments
Explanation - Golden Gate works on linear DNA fragments with specific overhangs; this is an advantage, not a disadvantage.
Correct answer is: Requires linear DNA fragments
Q.82 In Gibson Assembly, which enzyme provides the 5' exonuclease activity?
T5 exonuclease
T4 DNA ligase
Phusion DNA polymerase
EcoRI
Explanation - T5 exonuclease chews back DNA ends to create overlapping single-stranded regions.
Correct answer is: T5 exonuclease
Q.83 Which of the following plasmids is commonly used as a shuttle vector between E. coli and yeast?
pUC19
pRS plasmids
pET-28a
pGEX-4T-1
Explanation - pRS vectors contain both an E. coli origin and a yeast centromere, enabling replication in both hosts.
Correct answer is: pRS plasmids
Q.84 Which enzyme is used to 'methylate' plasmid DNA to protect it from restriction digestion in the host cell?
DNA methyltransferase
T4 DNA ligase
Taq polymerase
EcoRI
Explanation - Methyltransferases add methyl groups to DNA, protecting it from restriction enzymes that target unmethylated sites.
Correct answer is: DNA methyltransferase
Q.85 What is the purpose of a 'selection marker' that confers resistance to chloramphenicol?
To ensure plasmid replication
To kill cells that lack the plasmid
To provide a fluorescent signal
To increase plasmid copy number
Explanation - Chloramphenicol resistance allows growth only of cells that carry the plasmid.
Correct answer is: To kill cells that lack the plasmid
Q.86 Which of the following is a key advantage of using a 'low-copy number' plasmid for cloning?
Higher plasmid yield
Lower metabolic burden on host cells
Higher plasmid stability
Simpler purification
Explanation - Low-copy plasmids reduce the amount of plasmid DNA per cell, minimizing toxicity.
Correct answer is: Lower metabolic burden on host cells
Q.87 What does the term 'vector backbone' refer to in plasmid design?
The segment containing the gene of interest
The minimal set of sequences required for plasmid maintenance and selection
The promoter region
The antibiotic resistance gene
Explanation - The backbone provides origin of replication, selection marker, and other necessary elements.
Correct answer is: The minimal set of sequences required for plasmid maintenance and selection
Q.88 Which cloning method uses 'homologous recombination' to integrate DNA fragments into a plasmid vector?
Gibson Assembly
Red/ET recombination
TOPO cloning
Restriction enzyme cloning
Explanation - Red/ET uses recombination proteins to insert DNA fragments without restriction sites.
Correct answer is: Red/ET recombination
Q.89 Which of the following vectors is commonly used for the expression of secreted proteins in mammalian cells?
pET-28a
pcDNA3.1
pUC19
pGEM-T Easy
Explanation - pcDNA3.1 contains a CMV promoter and secretion signal sequences suitable for mammalian expression.
Correct answer is: pcDNA3.1
Q.90 What does the abbreviation 'TOPO' stand for in TOPO cloning?
Topological
Topoisomerase
Topological cloning
Topologically oriented
Explanation - TOPO cloning uses topoisomerase I to ligate PCR products directly into the vector.
Correct answer is: Topoisomerase
Q.91 Which enzyme is used to create 'sticky ends' from a blunt-ended DNA fragment before ligation?
T4 DNA ligase
T4 DNA polymerase
T4 DNA ligase
Taq polymerase
Explanation - T4 DNA polymerase has 3'→5' exonuclease activity that can create sticky ends.
Correct answer is: T4 DNA polymerase
Q.92 Which cloning strategy uses a 'self-ligating' plasmid that contains a blunt-end cutter and a ligase for seamless cloning?
TOPO cloning
Gibson Assembly
Ligase Cycling Reaction
PCR-based cloning
Explanation - TOPO cloning utilizes topoisomerase I to ligate blunt-ended PCR products without the need for restriction enzymes or ligase.
Correct answer is: TOPO cloning
Q.93 What is the purpose of a 'polyadenylation signal' in a mammalian expression vector?
To initiate transcription
To terminate transcription and stabilize mRNA
To provide antibiotic selection
To enhance plasmid replication
Explanation - The polyA signal signals the addition of a poly(A) tail, improving mRNA stability and export.
Correct answer is: To terminate transcription and stabilize mRNA
Q.94 Which of the following plasmid vectors is best suited for high-yield recombinant protein expression in E. coli?
pGEM-T Easy
pET-28a
pUC19
pBR322
Explanation - pET-28a contains a T7 promoter and His-tag for high-level expression and purification.
Correct answer is: pET-28a
Q.95 Which enzyme is used in the 'Golden Gate Assembly' technique to create seamless junctions?
T4 DNA ligase
Restriction endonuclease
T5 exonuclease
DNA polymerase I
Explanation - T4 DNA ligase seals nicks in the assembled DNA fragments during Golden Gate Assembly.
Correct answer is: T4 DNA ligase
Q.96 Which of the following is NOT a typical feature of a bacterial artificial chromosome (BAC)?
Large insert capacity (~300 kb)
Low copy number
Bacterial origin of replication
Yeast centromere
Explanation - BACs replicate in bacteria; they do not contain yeast centromeres.
Correct answer is: Yeast centromere
Q.97 What is the main purpose of a 'destination vector' in Gateway cloning?
To carry the gene of interest for expression
To enable restriction digestion
To provide antibiotic resistance only
To facilitate PCR amplification
Explanation - Destination vectors accept the recombined gene for subsequent expression or analysis.
Correct answer is: To carry the gene of interest for expression
Q.98 Which of the following is a characteristic of a 'suicide plasmid'?
It cannot replicate in the donor cell
It confers antibiotic resistance
It has a high copy number
It carries a fluorescent marker
Explanation - Suicide plasmids lack the origin of replication needed in the donor, forcing their transfer to a new host.
Correct answer is: It cannot replicate in the donor cell
Q.99 Which of the following enzymes is commonly used to 'close' nicks in double-stranded DNA during cloning?
DNA ligase
Taq polymerase
RNA polymerase
DNA helicase
Explanation - DNA ligase seals nicks in the phosphodiester backbone, producing continuous double-stranded DNA.
Correct answer is: DNA ligase
Q.100 What is the function of a 'promoter' in a plasmid vector?
To provide antibiotic resistance
To initiate transcription of the inserted gene
To prevent recombination
To provide a fluorescent tag
Explanation - A promoter recruits RNA polymerase to start transcription of the gene of interest.
Correct answer is: To initiate transcription of the inserted gene
Q.101 In a cloning experiment, why might you use a 'blue-white screening' assay?
To select for antibiotic resistance
To identify colonies that have successfully incorporated the insert
To measure plasmid copy number
To ensure plasmid replication
Explanation - Blue colonies indicate intact lacZ, while white colonies indicate disruption by an insert.
Correct answer is: To identify colonies that have successfully incorporated the insert
Q.102 Which enzyme is responsible for the removal of the 5' phosphate in the 'T4 DNA ligase' reaction?
Phosphatase
Kinase
Ligase
Exonuclease
Explanation - T4 DNA ligase uses ATP to form phosphodiester bonds; it does not remove phosphates.
Correct answer is: Ligase
Q.103 What is the main advantage of using a 'high-copy number' plasmid for protein expression?
Higher yield of recombinant protein
Increased plasmid stability
Reduced metabolic burden
Simplified plasmid purification
Explanation - High-copy plasmids produce many copies per cell, leading to increased expression of the cloned gene.
Correct answer is: Higher yield of recombinant protein
Q.104 Which of the following is a common selectable marker used in mammalian expression vectors?
Ampicillin resistance
Neomycin resistance
Kanamycin resistance
Chloramphenicol resistance
Explanation - The neomycin phosphotransferase gene (Neo) confers resistance to G418, used in mammalian cell selection.
Correct answer is: Neomycin resistance
Q.105 What is the role of the 'origin of replication (ori)' in a plasmid?
To initiate transcription
To confer antibiotic resistance
To allow replication of the plasmid in the host cell
To provide a fluorescent tag
Explanation - The ori is the sequence recognized by host replication machinery for plasmid duplication.
Correct answer is: To allow replication of the plasmid in the host cell
Q.106 Which of the following plasmid vectors is known for having an 'MCS' and an 'AmpR' gene?
pGEM-T Easy
pUC19
pBR322
pET-28a
Explanation - pUC19 contains a multiple cloning site and ampicillin resistance for selection.
Correct answer is: pUC19
Q.107 Which cloning strategy uses a 'catalytically inactive' version of a restriction enzyme to facilitate insertion?
In-Fusion cloning
TA cloning
Restriction enzyme cloning
Gibson Assembly
Explanation - In-Fusion cloning uses an enzyme that joins overlapping DNA fragments without the need for restriction digestion.
Correct answer is: In-Fusion cloning
Q.108 Which enzyme is used to 'methylate' plasmid DNA to protect it from restriction digestion in the host cell?
DNA methyltransferase
T4 DNA ligase
Taq polymerase
EcoRI
Explanation - DNA methyltransferases add methyl groups to DNA, protecting it from restriction enzymes that target unmethylated sites.
Correct answer is: DNA methyltransferase
Q.109 What is the purpose of a 'polyA tail signal' in a mammalian expression vector?
To start transcription
To terminate transcription and stabilize mRNA
To provide antibiotic resistance
To increase plasmid copy number
Explanation - The polyA signal signals the addition of a poly(A) tail, improving mRNA stability and export.
Correct answer is: To terminate transcription and stabilize mRNA
Q.110 Which of the following plasmid vectors is commonly used for the expression of secreted proteins in mammalian cells?
pET-28a
pcDNA3.1
pUC19
pGEM-T Easy
Explanation - pcDNA3.1 contains a CMV promoter and secretion signal sequences suitable for mammalian expression.
Correct answer is: pcDNA3.1
Q.111 What is the role of the 'multiple cloning site (MCS)' in a plasmid vector?
To provide a promoter
To provide antibiotic selection
To allow insertion of DNA using various restriction enzymes
To encode a fluorescent protein
Explanation - The MCS contains several unique restriction sites for flexible cloning.
Correct answer is: To allow insertion of DNA using various restriction enzymes
Q.112 Which of the following is NOT a typical selection marker used in mammalian expression vectors?
Neomycin (G418)
Puromycin
Ampicillin
Blasticidin
Explanation - Ampicillin is used for bacterial selection; mammalian vectors use antibiotic markers like G418.
Correct answer is: Ampicillin
Q.113 Which of the following enzymes is used for the 'T7 RNA polymerase' transcription of a plasmid carrying a T7 promoter?
T7 RNA polymerase
T4 DNA ligase
Taq polymerase
Reverse transcriptase
Explanation - T7 RNA polymerase recognizes the T7 promoter and transcribes the downstream gene.
Correct answer is: T7 RNA polymerase